The liberation of calcium from in intracellular stores into the cytosol is used as a signaling mechanism by virtually all cell types to regulate functions as diverse as secretion, contraction, proliferation and cell death. Improved imaging technology has revealed that calcium liberation through pathways involving both inositol trisphosphate and ryanodine receptor/channels occurs discontinuously, as 'elementary' calcium release events. These transient, localized, subcellular free [Ca2+] elevations arise at clusters of channels that form discrete functional release sites within the endoplasmic reticulum. Individual sites can generate autonomous events involving single or multiple channels, and their activity may be coordinated by calcium diffusion and calcium-induced calcium release to propagate global cellular calcium waves. Elementary events thus form the basic building blocks underlying the complex spatiotemporal calcium signals that permit graded and selective regulation of cell functions. An understanding of their generation, interaction and functional consequences is, therefore, pivotal to understand the physiological and pathological functioning of the ubiquitous calcium messenger pathway. Our specific aims are to determine, (i) the mechanisms underlying the generation of elementary calcium events, (h) the coordination between events allowing the initiation and propagation of calcium waves, and (iii) the principles underlying specific activation of effector systems by local calcium microdomains. We will use Xenopus oocytes and cardiac myocytes as model systems to study signaling by inositol trisphosphate- and ryanodine-receptors, respectively. Furthermore, use of cultured cell lines will allow investigation of events generated by other receptor isoforms. Our experimental methodology involves 1- and 2-photon confocal microscopy with high (less than 0.5 mum and 1 ms) resolution to image subcellular calcium events in intact cells evoked by photorelease of inositol trisphosphate and Ca2+. These optical techniques provide a unique opportunity to visualize single channel activity in living cells, and our overall goal is to elucidate how individual calcium release channels contribute to cellular calcium responses.